TITLE: The RNA nanomachines of gene expression dissected at the single molecule level ABSTRACT: Over two decades, the Walter lab has contributed to the RNA field by building a broad research portfolio focused on dissecting the mechanisms of the nanoscale RNA machines of gene expression ? ranging from small viroidal ribozymes and bacterial riboswitches to the eukaryotic spliceosome ? by single molecule fluorescence microscopy. Leveraging this expertise, the two long-term goals of the current proposal are to: 1.) Apply our established mechanistic enzymology approaches to an ever broader set of RNAs involved in regulating transcription, translation and splicing, seizing the opportunities arising from the continuing discoveries of new functional RNAs. 2.) Push the limits of our approaches to be able to probe increasingly complex biological contexts and mechanisms since unexpected discoveries ? as we found ? often await where individual RNA nanomachines interact. In pursuit of these goals, we will address the overarching hypothesis that dynamic RNA structures are a major determinant of the outcomes of gene expression, often in ways that have been overlooked by a field that historically was rooted in genetics, where genes regularly were drawn as rectangular boxes, and function commonly was thought of as dictated by sequence rather than structure. Such thinking is countered by, for example, the fact that nascent RNA structure has a significant impact on transcription in the form of regulatory riboswitches embedded near the 5' ends of bacterial mRNAs and of transcription terminator hairpins at the 3' end. Conversely, the time-ordered, 5'-to-3' directional RNA synthesis of transcription often yields kinetically trapped RNA folds distinct from the most thermodynamically stable structure of a refolded full-length transcript. Encapsulating the power of our pursuit, we recently combined single-molecule, biochemical and computational simulation approaches to show that transcriptional pausing at a site immediately downstream of a riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced consensus pause sequence, and electrostatic and steric interactions with the exit channel of bacterial RNA polymerase. We posit that many more examples of such intimate structural and kinetic coupling between RNA folding and gene expression remain to be discovered, leading to the exquisite regulatory control and kinetic proofreading enabling all life processes. To reveal more such couplings, we will probe the dynamics of carefully purified transcriptional and translational riboswitch-containing, as well as spliceosomal, gene expression complexes using a tailored combination of single molecule fluorescence resonance energy transfer (smFRET), Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) based on super-resolved co-localization of RNA targets and fluorescent probes, cryo-electron microscopy ? augmented by a proposed dye-based single molecule correlative light electron microscopy (smCLEM) ? and, where appropriate, molecular dynamics simulations. We anticipate that these studies have the potential to transform our understanding of RNA structure-function relationships in general, and of how RNA structure is governing the function of cellular gene expression machines in particular.